Highlights Translation 3

1. Post-translational (= after translation has occurred) processing of proteins gives them their final structure and properties. One such modification was discussed for insulin. This involves proteolytic cleavage at two places in the insulin precursor, giving rise to a final structure that has two chains held together by disulfide bonds.

2. Proteins do not have long lifetimes in the cell. They are broken down in cells in cellular structures called proteasomes. Proteins targeted for degradation are tagged with the peptide known as ubiquitin.

3. Selenocysteine is a rare amino acid occasionally incorporated into proteins. It is sometimes called the 21st amino acid. It is the only modified amino acid in proteins that gets there by direct incorporation rather than post-translational modification. It is because of this that trace amounts of selenium are needed in the diet.

4. Proper folding of proteins is important. A complex in E. coli that facilitates proper folding of proteins is the GroES/GroEL complex, which provides a chamber for a protein to fold in without interference of other proteins during the folding process.

Biotechnology

1. Biotechnology employs knowledge of the processes of DNA replication, transcription, and translation to make useful products.

2. Cloning of organisms is one application of biotechnology.

3. The modern era of biotechnology began with the discovery of restriction endonucleases (=restriction enzymes). These are enzymes isolated from bacterial cells that bind to DNA, recognize a specific DNA sequence, and cut at that sequence.

4. Many restriction enzymes recognize specific inverted repeat sequences and cut there. As shown for EcoRI, the cut DNA fragments have overhanging ends called "sticky" that can be used to form base pairs that are useful for putting pieces back together with DNA ligase.

5. Restriction enzymes are bacterial defense systems. In normal bacterial cells, they are paired with an enzyme called a methylase. The function of the methylase is the put a methyl group on the sequence the restriction enzyme would otherwise cut. Cellular DNA is protected in this way. Invading virus sequences, however, aren't methylated, so their DNAs get cut by the restriction enzymes.

6. If one piece of a DNA molecule is linked to a foreign DNA molecule, a recombinant molecule is created. If one uses DNA ligase to link a DNA to a circular DNA that replicates in bacteria (called a plasmid), then the foreign DNA will be replicated in the bacterial cells. If the plasmid has a promoter that can control transcription, the plasmid is known as an expression vector. An expression vector provides a means of using bacteria to transcribe and translate foreign genes.

7. The effciency with which plasmids can be put into cells is about 1 cell in 100000 at the best. Antibiotic resistance (carried in a gene on the plasmid) is a good way of screening for bacterial cells that get a plasmid. This is done by plating bacteria on a plate of agar that contains a specific antibiotic that the plasmid gene gives resistance to. Only cells with the plasmid will grow.

9. When recombinants are made by ligating a foreign DNA into a plasmid, this process too is not very efficient. A method for determining not only which cells get plasmids, but also which cells get plasmids with DNA inserted into them is also desirable. This latter method involves what is called blue-white screeening and it employs the lac-z (beta-galactosidase) gene of E. coli.

10. In blue-white screening, a plasmid contains the following things - antibiotic resistance, origin or replication, and lac-z gene with at least one restriction site in the gene. Researchers cut DNA with the same restriction enzyme as the restriction enzyme site in lac-Z and then ligate the fragments in the presence of the plasmid cut with the same gene. If a plasmid gets an insert into the lac-z gene, the gene doesn't function. If no insert goes into the gene and the plasmid simply re-forms the original gene, lac-z functions.

11. The ligation mixture is transformed into E. coli cells and plated on an antibiotic (to ensure that only cells with plasmids grow) and a compound known as X-gal. X-gal has the property that if the lac-z gene is present, it will cleave it and produce a blue color. If no lac-Z is present, a white color is produced. Thus, researchers can tell which cells have a plasmid with an insert by looking for which colonies are white. Blue colonies come from cells that plasmids with no insert (functional lac-Z gene), whereas white colonies come from cells with plasmids with inserts that interrupt the lac-Z gene (no functional lac-Z gene).